The emergence of Enterococcus faecalis as a significant human pathogen, with many strains becoming resistant to antibiotics emphasizes the need to develop new and effective means of controlling infections by these organisms. An alternative approach to antibiotics for treating E. faecalis infections is the use of E. faecalis-specific bacterial viruses (bacteriophages) that can lyse and kill cells of these bacteria in the course of a normal lytic infective cycle. To be usable for bacteriophage therapy a bacterial virus must be free of any undesirable genes, such as genes for toxins or antibiotic resistance, and not be capable of lysogeny, an infection pathway of temperate bacteriophages where the viral DNA remains largely dormant in the infected cell, either as an integrated unit within the host chromosome or as a self-replicating extra-chromosomal genetic element within the cell. Recently, we isolated a bacteriophage, which infects strains of E. faecalis. Our sequencing and analysis of the bacteriophage DNA found it to be free of any detectable toxin-related genes, however, as a temperate bacteriophage, it would not be suitable as a therapeutic agent in its wild type state. To rectify this limitation, we plan to genetically engineer a virulent derivative of this virus that would be incapable of lysogeny. We will accomplish this by synthesizing a synthetic viral genome in which lysogeny-related genes are either deleted or mutated. In effect, we will custom design a viral genome to produce a synthetic virus that is optimally suited to function as a therapeutic antimicrobial agent. The synthetic virus that we will generate in this project will be screened for antimicrobial effectiveness against E. faecalis. PUBLIC HEALTH RELEVANCE: Enterococcus faecalis infections are becoming a major health problem, due to the increasing frequency of antibiotic resistance among strains of this organism. In this application, we propose to use synthetic genome technology to generate a derivative of bacteriophage phiEf11, a bacterial virus that we have previously isolated that will be an effective antimicrobial agent for treating these infections.